Conserved N-terminal Regulation of the ACA8 Calcium Pump with Two Calmodulin Binding Sites

IF 4.7 2区生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Journal of Molecular Biology Pub Date : 2024-08-20 DOI:10.1016/j.jmb.2024.168747

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Abstract

The autoinhibited plasma membrane calcium ATPase ACA8 from A. thaliana has an N-terminal autoinhibitory domain. Binding of calcium-loaded calmodulin at two sites located at residues 42–62 and 74–96 relieves autoinhibition of ACA8 activity.

Through activity studies and a yeast complementation assay we investigated wild-type (WT) and N-terminally truncated ACA8 constructs (Δ20, Δ30, Δ35, Δ37, Δ40, Δ74 and Δ100) to explore the role of conserved motifs in the N-terminal segment preceding the calmodulin binding sites. Furthermore, we purified WT, Δ20- and Δ100-ACA8, tested activity in vitro and performed structural studies of purified Δ20-ACA8 stabilized in a lipid nanodisc to explore the mechanism of autoinhibition.

We show that an N-terminal segment between residues 20 and 35 including conserved Phe32, upstream of the calmodulin binding sites, is important for autoinhibition and the activation by calmodulin. Cryo-EM structure determination at 3.3 Å resolution of a beryllium fluoride inhibited E2 form, and at low resolution for an E1 state combined with AlphaFold prediction provide a model for autoinhibition, consistent with the mutational studies.

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具有两个钙调蛋白结合位点的 ACA8 钙泵 N 端保守调控。

拟南芥质膜钙离子ATP酶ACA8具有一个N末端的自动抑制结构域。钙载钙调蛋白与位于 42-62 和 74-96 残基的两个位点的结合可解除对 ACA8 活性的自动抑制。通过活性研究和酵母互补试验，我们研究了野生型（WT）和 N 端截短的 ACA8 构建物（Δ20、Δ30、Δ35、Δ37、Δ40、Δ74 和 Δ100），以探索钙调蛋白结合位点之前的 N 端片段中保守基团的作用。此外，我们纯化了WT、Δ20和Δ100-ACA8，在体外测试了其活性，并对稳定在脂质纳米盘中的纯化Δ20-ACA8进行了结构研究，以探索其自动抑制机制。我们的研究表明，残基 20 和 35 之间的 N 端片段（包括钙调蛋白结合位点上游的保守 Phe32）对于钙调蛋白的自动抑制和激活非常重要。以 3.3 Å 分辨率测定的氟化铍抑制 E2 状态的低温电子显微镜结构和低分辨率测定的 E1 状态的低温电子显微镜结构与 AlphaFold 预测相结合，提供了一个与突变研究相一致的自动抑制模型。

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来源期刊

Journal of Molecular Biology 生物-生化与分子生物学

CiteScore

11.30

自引率

1.80%

发文量

412

审稿时长

28 days

期刊介绍： Journal of Molecular Biology (JMB) provides high quality, comprehensive and broad coverage in all areas of molecular biology. The journal publishes original scientific research papers that provide mechanistic and functional insights and report a significant advance to the field. The journal encourages the submission of multidisciplinary studies that use complementary experimental and computational approaches to address challenging biological questions. Research areas include but are not limited to: Biomolecular interactions, signaling networks, systems biology; Cell cycle, cell growth, cell differentiation; Cell death, autophagy; Cell signaling and regulation; Chemical biology; Computational biology, in combination with experimental studies; DNA replication, repair, and recombination; Development, regenerative biology, mechanistic and functional studies of stem cells; Epigenetics, chromatin structure and function; Gene expression; Membrane processes, cell surface proteins and cell-cell interactions; Methodological advances, both experimental and theoretical, including databases; Microbiology, virology, and interactions with the host or environment; Microbiota mechanistic and functional studies; Nuclear organization; Post-translational modifications, proteomics; Processing and function of biologically important macromolecules and complexes; Molecular basis of disease; RNA processing, structure and functions of non-coding RNAs, transcription; Sorting, spatiotemporal organization, trafficking; Structural biology; Synthetic biology; Translation, protein folding, chaperones, protein degradation and quality control.